Cell Cycle Lengthening Research Articles

Temporal and spatial transcriptomic dynamics across brain development in Xenopus laevis tadpoles.

Amphibian metamorphosis is a transitional period that involves significant changes in the cell-type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing Xenopus laevis tadpole brain during this period in two ways: first, over stages of the development in the midbrain and, second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that different programs may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell-type and biological pathway dynamics in the brain during metamorphosis.

Trp53 ablation fails to prevent microcephaly in mouse pallium with impaired minor intron splicing.

Minor spliceosome inhibition due to mutations in RNU4ATAC are linked to primary microcephaly. Ablation of Rnu11, which encodes a minor spliceosome snRNA, inhibits the minor spliceosome in the developing mouse pallium, causing microcephaly. There, cell cycle defects and p53-mediated apoptosis in response to DNA damage resulted in loss of radial glial cells (RGCs), underpinning microcephaly. Here, we ablated Trp53 to block cell death in Rnu11 cKO mice. We report that Trp53 ablation failed to prevent microcephaly in these double knockout (dKO) mice. We show that the transcriptome of the dKO pallium was more similar to the control compared with the Rnu11 cKO. We find aberrant minor intron splicing in minor intron-containing genes involved in cell cycle regulation, resulting in more severely impaired mitotic progression and cell cycle lengthening of RGCs in the dKO that was detected earlier than in the Rnu11 cKO. Furthermore, we discover a potential role of p53 in causing DNA damage in the developing pallium, as detection of γH2aX+ was delayed in the dKO. Thus, we postulate that microcephaly in minor spliceosome-related diseases is primarily caused by cell cycle defects.

Proliferation rate and differentiation potential are independent during the transition from neurogenesis to gliogenesis in the mouse embryonic spinal cord.

Neural stem cells (NSC) restrict their differentiation potential as the central nervous system develops. Experimental evidence suggests that the mechanisms governing the transition from the neurogenic to the gliogenic phase irreversibly affect the ability of NSC to generate neurons. Cell cycle regulation has been associated with cell fate in different models. In this work, we assessed the temporal correlation between the loss of the neurogenic potential and cell cycle lengthening of NSC obtained from embryonic mouse spinal cords, during the transition of the neurogenic to the gliogenic phase, using neurospheres. We also used the cell cycle inhibitor Olomoucine to increase cell cycle length by decreasing the proliferation rate. Our results show that neurospheres obtained from a neurogenic stage give rise mostly to neurons, whereas those obtained from later stages produce preferentially glial cells. During the transition from neurogenesis to gliogenesis, the proliferation rate dropped, and the cell cycle length increased 1.5 folds, as monitored by DNA BrdU incorporation. Interestingly, Olomoucine-treated neurogenic-neurospheres display a reduced proliferation rate and preserve their neurogenic potential. Our results suggest that the mechanisms that restrict the differentiation potential of NSC are independent of the proliferation control.

Let-7 regulates cell cycle dynamics in the developing cerebralcortex and retina

In the neural progenitors of the developing central nervous system(CNS), cell proliferation is tightly controlled and coordinated with cell fatedecisions. Progenitors divide rapidly during early development and their cell cyclelengthens progressively as development advances to eventually give rise to a tissueof the correct size and cellular composition. However, our understanding of themolecules linking cell cycle progression to developmental time is incomplete. Here,we show that the microRNA (miRNA) let-7 accumulates in neural progenitors over timethroughout the developing CNS. Intriguingly, we find that the level and activity oflet-7 oscillate as neural progenitors progress through the cell cycle by in situ hybridization and fluorescent miRNA sensoranalyses. We also show that let-7 mediates cell cycle dynamics: increasing the levelof let-7 promotes cell cycle exit and lengthens the S/G2 phase of the cell cycle,while let-7 knock down shortens the cell cycle in neural progenitors. Together, ourfindings suggest that let-7 may link cell proliferation to developmental time andregulate the progressive cell cycle lengthening that occurs duringdevelopment.

Both Nuclear Size and DNA Amount Contribute to Midblastula Transition Timing in Xenopus laevis

During early Xenopus laevis embryogenesis both nuclear and cell volumes decrease with the nuclear-to-cytoplasmic (N/C) volume ratio reaching a maximum at the midblastula transition (MBT). At the MBT, embryonic transcription is upregulated and cell cycles lengthen. Early studies demonstrated a role for the DNA-to-cytoplasmic ratio in the control of MBT timing. By altering nuclear size, we previously showed that the N/C volume ratio also contributes to proper MBT timing. Here we examine the relative contributions of nuclear size and DNA amount to MBT timing by simultaneously altering nuclear size and ploidy in X. laevis embryos. Compared to diploid embryos, haploids exhibited a delay in both zygotic gene expression and cell cycle lengthening, while diploid embryos with increased N/C volume ratios showed early expression of zygotic genes and premature lengthening of cell cycles. Interestingly, haploids with increased N/C volume ratios exhibited an intermediate effect on the timing of zygotic gene expression and cell cycle lengthening. Decreasing nuclear size in post-MBT haploid embryos caused a further delay in cell cycle lengthening and the expression of some zygotic genes. Our data suggest that both the N/C volume ratio and DNA amount contribute to the regulation of MBT timing with neither parameter being dominant.

Ploidy has little effect on timing early embryonic events in the haplo-diploid wasp Nasonia.

The nucleocytoplasmic (N/C) ratio plays a prominent role in the maternal-to-zygotic transition (MZT) in many animals. The effect of the N/C ratio on cell-cycle lengthening and zygotic genome activation (ZGA) has been studied extensively in Drosophila, where haploid embryos experience an additional division prior to completing cellularization and triploid embryos cellularize precociously by one division. In this study, we set out to understand how the obligate difference in ploidy in the haplodiploid wasp, Nasonia, affects the MZT and which aspects of the Drosophila MZT are conserved. While subtle differences in early embryonic development were observed in comparisons among haploid, diploid, and triploid embryos, in all cases embryos cellularize at cell cycle 12. When ZGA was inhibited, both diploid female, and haploid male, embryos went through 12 syncytial divisions and failed to cellularize before dying without further divisions. We also found that key players of the Drosophila MZT are conserved in Nasonia but have novel expression patterns. Our results suggest that zygotically expressed genes have a reduced role in determining the timing of cellularization in Nasonia relative to Drosophila, and that a stronger reliance on a maternal timer is more compatible with species where variations in embryonic ploidy are obligatory.

Cyclin A2 promotes DNA repair in the brain during both development and aging.

Various stem cell niches of the brain have differential requirements for Cyclin A2. Cyclin A2 loss results in marked cerebellar dysmorphia, whereas forebrain growth is retarded during early embryonic development yet achieves normal size at birth. To understand the differential requirements of distinct brain regions for Cyclin A2, we utilized neuroanatomical, transgenic mouse, and mathematical modeling techniques to generate testable hypotheses that provide insight into how Cyclin A2 loss results in compensatory forebrain growth during late embryonic development. Using unbiased measurements of the forebrain stem cell niche, we parameterized a mathematical model whereby logistic growth instructs progenitor cells as to the cell-types of their progeny. Our data was consistent with prior findings that progenitors proliferate along an auto-inhibitory growth curve. The growth retardation in CCNA2-null brains corresponded to cell cycle lengthening, imposing a developmental delay. We hypothesized that Cyclin A2 regulates DNA repair and that CCNA2-null progenitors thus experienced lengthened cell cycle. We demonstrate that CCNA2-null progenitors suffer abnormal DNA repair, and implicate Cyclin A2 in double-strand break repair. Cyclin A2's DNA repair functions are conserved among cell lines, neural progenitors, and hippocampal neurons. We further demonstrate that neuronal CCNA2 ablation results in learning and memory deficits in aged mice.

Arsenic-induced S phase cell cycle lengthening is associated with ROS generation, p53 signaling and CDC25A expression

Cellular response to arsenic is strongly dependent on p53 functional status. Primarily arresting the cell cycle in G1 or G2/M phases, arsenic treatment also induces an increase in the S-phase time in wild-type p53 cells. In contrast, cells with a non-functional p53 display only a subtle increase in the S phase, indicating arsenic differentially affects the cell cycle depending on p53 status. Importantly, it has been reported that arsenic induces reactive oxygen species (ROS), a process counteracted by p53. To evaluate the participation of p53 in the lengthening of the S phase and the connection between the transient cell cycle arrest and oxidative stress, we evaluated the cell response to arsenic in MCF-7 and H1299 cells, and analyzed p53’s role as a transcription factor in regulating genes involved in ROS reduction and S phase transition. Herein, we discovered that arsenic induced an increase in the population of S phase cells that was dependent on the presence and transcriptional activity of p53. Furthermore, for the first time, we demonstrate that arsenic activates p53-dependent transcription of ROS detoxification genes, such as SESN1, and by an indirect mechanism involving ATF3, genes that could be responsible for the S phase cell cycle arrest, such as CDC25A.

Spindle assembly checkpoint acquisition at the mid-blastula transition.

The spindle assembly checkpoint (SAC) maintains the fidelity of chromosome segregation during mitosis. Nonpathogenic cells lacking the SAC are typically only found in cleavage stage metazoan embryos, which do not acquire functional checkpoints until the mid-blastula transition (MBT). It is unclear how proper SAC function is acquired at the MBT, though several models exist. First, SAC acquisition could rely on transcriptional activity, which increases dramatically at the MBT. Embryogenesis prior to the MBT relies primarily on maternally loaded transcripts, and if SAC signaling components are not maternally supplied, the SAC would depend on zygotic transcription at the MBT. Second, checkpoint acquisition could depend on the Chk1 kinase, which is activated at the MBT to elongate cell cycles and is required for the SAC in somatic cells. Third, SAC function could depend on a threshold nuclear to cytoplasmic (N:C) ratio, which increases during pre-MBT cleavage cycles and dictates several MBT events like zygotic transcription and cell cycle remodeling. Finally, the SAC could by regulated by a timer mechanism that coincides with other MBT events but is independent of them. Using zebrafish embryos we show that SAC acquisition at the MBT is independent of zygotic transcription, indicating that the checkpoint program is maternally supplied. Additionally, by precociously lengthening cleavage cycles with exogenous Chk1 activity, we show that cell cycle lengthening and Chk1 activity are not sufficient for SAC acquisition. Furthermore, we find that SAC acquisition can be uncoupled from the N:C ratio. Together, our findings indicate that SAC acquisition is regulated by a maternally programmed developmental timer.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

Macrophage and T-cell fate control: insights from tracking transcription factor dynamics in single cells

Hematopoietic stem and progenitor cells employ circuits of regulatory genes to integrate developmental signals and stabilize fate choices. These progenitors show considerable cell-to-cell heterogeneity in their fate choices; thus, to better understand how regulatory genes control fate decisions, we tracked their levels in single progenitors over time using timelapse live-cell imaging. We examined two hematopoietic fate transitions: 1) macrophage differentiation, driven by the up-regulation of the myeloid transcription factor PU.1; and 2) T-cell fate commitment, controlled by the activation of the T-cell specific transcription factor Bcl11b. In our study of macrophage differentiation, we found that cell cycle length acts as critical mediator in the positive feedback circuit controlling differentiation (Kueh et al. 2013). By following PU.1 regulation in single cells containing a knock-in PU.1-GFP reporter, we found that developing macrophages lengthen their cell cycles to promote stable PU.1 accumulation, and that PU.1 itself promotes cell cycle lengthening, completing a positive feedback loop that stabilizes its own expression. Mathematical modeling furthered showed that this cell cycle feedback circuit robustly stabilizes a slow-dividing differentiated state. In our studies on T-cell fate commitment, we found that Notch signaling – the primary driver of development – enhances the frequency of all-or-none Bcl11b gene activation to promote commitment. By analyzing progenitors from mice containing a knock-in Bcl11b-YFP reporter, we found that uncommitted (Bcl11b-DN2A) progenitors can activate Bcl11b transcription and undergo fate commitment in the absence of Notch signaling, and that Notch signaling does not modulate the level of Bcl11b transcription, but instead increases the rate at which progenitors switch Bcl11b to an actively expressing state. These results reveal insights into how signaling pathways activate regulatory gene expression to instruct cell fate.

Cell cycle regulation of proliferation versus differentiation in the central nervous system.

Formation of the central nervous system requires a period of extensive progenitor cell proliferation, accompanied or closely followed by differentiation; the balance between these two processes in various regions of the central nervous system gives rise to differential growth and cellular diversity. The correlation between cell cycle lengthening and differentiation has been reported across several types of cell lineage and from diverse model organisms, both in vivo and in vitro. Furthermore, different cell fates might be determined during different phases of the preceding cell cycle, indicating direct cell cycle influences on both early lineage commitment and terminal cell fate decisions. Significant advances have been made in the last decade and have revealed multi-directional interactions between the molecular machinery regulating the processes of cell proliferation and neuronal differentiation. Here, we first introduce the modes of proliferation in neural progenitor cells and summarise evidence linking cell cycle length and neuronal differentiation. Second, we describe the manner in which components of the cell cycle machinery can have additional and, sometimes, cell-cycle-independent roles in directly regulating neurogenesis. Finally, we discuss the way that differentiation factors, such as proneural bHLH proteins, can promote either progenitor maintenance or differentiation according to the cellular environment. These intricate connections contribute to precise coordination and the ultimate division versus differentiation decision.

Trrap-Dependent Histone Acetylation Specifically Regulates Cell-Cycle Gene Transcription to Control Neural Progenitor Fate Decisions

Fate decisions in neural progenitor cells are orchestrated via multiple pathways, and the role of histone acetylation in these decisions has been ascribed to a general function promoting gene activation. Here, we show that the histone acetyltransferase (HAT) cofactor transformation/transcription domain-associated protein (Trrap) specifically regulates activation of cell-cycle genes, thereby integrating discrete cell-intrinsic programs of cell-cycle progression and epigenetic regulation of gene transcription in order to control neurogenesis. Deletion of Trrap impairs recruitment of HATs and transcriptional machinery specifically to E2F cell-cycle target genes, disruptingtheir transcription with consequent cell-cycle lengthening specifically within cortical apical neural progenitors (APs). Consistently, Trrap conditional mutants exhibit microcephaly because of premature differentiation of APs into intermediate basal progenitors and neurons, and overexpressing cell-cycle regulators invivo can rescue these premature differentiation defects. These results demonstrate an essential and highly specific role for Trrap-mediated histone regulation in controlling cell-cycle progression and neurogenesis.

Stability of bacterial toggle switches is enhanced by cell-cycle lengthening by several orders of magnitude

Bistable regulatory elements are important for nongenetic inheritance, increase of cell-to-cell heterogeneity allowing adaptation, and robust responses at the population level. Here, we study computationally the bistable genetic toggle switch-a small regulatory network consisting of a pair of mutual repressors-in growing and dividing bacteria. We show that as cells with an inhibited growth exhibit high stability of toggle states, cell growth and divisions lead to a dramatic increase of toggling rates. The toggling rates were found to increase with rate of cell growth, and can be up to six orders of magnitude larger for fast growing cells than for cells with the inhibited growth. The effect is caused mainly by the increase of protein and mRNA burst sizes associated with the faster growth. The observation that fast growth dramatically destabilizes toggle states implies that rapidly growing cells may vigorously explore the epigenetic landscape enabling nongenetic evolution, while cells with inhibited growth adhere to the local optima. This can be a clever population strategy that allows the slow growing (but stress resistant) cells to survive long periods of unfavorable conditions. Simultaneously, at favorable conditions, this stress resistant (but slowly growing-or not growing) subpopulation may be replenished due to a high switching rate from the fast growing population.

Positive Feedback Between PU.1 and the Cell Cycle Controls Myeloid Differentiation

Regulatory gene circuits with positive-feedback loops control stem cell differentiation, but several mechanisms can contribute to positive feedback. Here, we dissect feedback mechanisms through which the transcription factor PU.1 controls lymphoid and myeloid differentiation. Quantitative live-cell imaging revealed that developing B cells decrease PU.1 levels by reducing PU.1 transcription, whereas developing macrophages increase PU.1 levels by lengthening their cell cycles, which causes stable PU.1 accumulation. Exogenous PU.1 expression in progenitors increases endogenous PU.1 levels by inducing cell cycle lengthening, implying positive feedback between a regulatory factor and the cell cycle. Mathematical modeling showed that this cell cycle-coupled feedback architecture effectively stabilizes a slow-dividing differentiated state. These results show that cell cycle duration functions as an integral part of a positive autoregulatory circuit to control cell fate.

Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation

Neurogenin 2 (Ngn2) controls neuronal differentiation cell-autonomously by transcriptional activation of targets such as NeuroD, while simultaneously controlling progenitor maintenance non-cell-autonomously by upregulating Delta expression and Notch signalling. Reduction in Cdk-dependent multisite phosphorylation of Ngn2 enhances its promoter binding affinity. This leads specifically to an increase in neuronal differentiation without an apparent increase in progenitor maintenance via Delta-Notch signalling, although the mechanism underlying this imbalance remains unclear. Here we show in Xenopus embryos and mouse P19 cells that the NeuroD promoter is substantially more sensitive to the phosphorylation status of Ngn2 than the Delta promoter, and that this can be attributed to differences in the ease of promoter activation. In addition, we also show that the phosphorylation status of Ngn2 regulates sensitivity to Notch signalling. These observations explain how Ngn2 post-translational modification in response to changes in the cell cycle kinase environment results in enhanced neuronal differentiation upon cell cycle lengthening.

Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis

During development of the central nervous system, the transition from progenitor maintenance to differentiation is directly triggered by a lengthening of the cell cycle that occurs as development progresses. However, the mechanistic basis of this regulation is unknown. The proneural transcription factor Neurogenin 2 (Ngn2) acts as a master regulator of neuronal differentiation. Here, we demonstrate that Ngn2 is phosphorylated on multiple serine-proline sites in response to rising cyclin-dependent kinase (cdk) levels. This multi-site phosphorylation results in quantitative inhibition of the ability of Ngn2 to induce neurogenesis in vivo and in vitro. Mechanistically, multi-site phosphorylation inhibits binding of Ngn2 to E box DNA, and inhibition of DNA binding depends on the number of phosphorylation sites available, quantitatively controlling promoter occupancy in a rheostat-like manner. Neuronal differentiation driven by a mutant of Ngn2 that cannot be phosphorylated by cdks is no longer inhibited by elevated cdk kinase levels. Additionally, phosphomutant Ngn2-driven neuronal differentiation shows a reduced requirement for the presence of cdk inhibitors. From these results, we propose a model whereby multi-site cdk-dependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development.

A Highly Conserved Poc1 Protein Characterized in Embryos of the Hydrozoan Clytia hemisphaerica: Localization and Functional Studies

Poc1 (Protein of Centriole 1) proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized “germ plasm” region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

Primate-specific RFPL1 gene controls cell-cycle progression through cyclin B1/Cdc2 degradation

Ret finger protein-like 1 (RFPL1) is a primate-specific target gene of Pax6, a key transcription factor for pancreas, eye and neocortex development. However, its cellular activity remains elusive. In this article, we report that Pax6-elicited expression of the human (h)RFPL1 gene in HeLa cells can be enhanced by in vivo p53 binding to its promoter and therefore investigated the hypothesis that hRFPL1 regulates cell-cycle progression. Upon expression in these cells, hRFPL1 decreased cell number through a kinase-dependent mechanism as PKC activates and Cdc2 inhibits hRFPL1 activity. hRFPL1 antiproliferative activity led to an increased cell population in G(2)/M phase and specific cyclin B1 and Cdc2 downregulations, which were precluded by a proteasome inhibitor. Specifically, cytoplasm-localized hRFPL1 prevented cyclin B1 and Cdc2 accumulation during interphase. Consequently, cells showed a delayed entry into mitosis and cell-cycle lengthening resulting from a threefold increase in G(2) phase duration. Given previous reports that RFPL1 is expressed during cell differentiation, its impact on cell-cycle lengthening therefore provides novel insights into primate-specific development.

Receptor Density Is Key to the Alpha2/Beta Interferon Differential Activities

Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.